NK-1, NK-3 and NK-4 are Drosophila homeobox genes which constitute a novel cluster at the 93E region of the third chromosome (Kim and Nirenberg, PNAS 86: 7716-7720, 1989). In situ hybridization showed that these genes are regulated temporally and spatially, and mostly expressed in embryonic mesodermal cells (NK-3 and NK-4) and specific muscle segments (NK-1) suggesting that these genes may be involved in mesodermal cell differentiation and/or muscle segment formation. In order to study the function of NK-homeobox genes at this locus, we initiated mutant screening by the enhancer trap method. Among 350 lines analyzed, we established 58 lines and obtained 11 3rd chromosome lethal lines as of this moment. As an alternative approach, we generated three artificial ribozymes targeting NK-4 mRNA, which appeared before gastrulation, and showed that these ribozymes are active in vitro. Analysis of enhancer trap lines and microinjection of artificial ribozymes into Drosophila embryos are in progress. To find cis-acting elements controlling the expression of NK-1, we analyzed 4 kb of the 5' upstream region of NK-1 using a transient expression system. In C2C12 mouse myoblast cells, a 420 bp DNA fragment (from -420 to +1), which is proximal to the NK-1 promoter, showed strong enhancer activity. We found that this region has a putative transcription factor MyoD binding site, suggesting the involvement of MyoD in muscle-specific expression of the NK-1 homeobox gene. We have cloned a new mouse NK-1 homeobox (NKx 1.1) genomic DNA (23 overlapping clones) and cDNAs (from an embryonic cDNA library), and sequenced 3 kb of NKx 1.1 genomic DNA. Sequence analysis showed direct repeat sequences (143 bp) in the 3' downstream region, and that the NKx 1.1 homeodomain has a 95% similarity to the Drosophila NK-1 homeodomain. RT-PCR analysis showed that the NKx 1.1 is expressed as early as embryonic day 10. We have also cloned and sequenced a novel Drosophila pou-homeobox gene, Dph 1. Characterization of Dph 1 is underway.